Vasculitis is a common pathological outcome in cocaine addicts, 80% of whom co-abuse cocaine and ethanol. We believe that the use of cocaethylene (CE, a conjugate of cocaine and ethanol, produced in vivo) in studies of cocaine abuse will accurately represent pathogenic sequelae. Our long-term goal is to better understand the cellular and subcellular mechanisms of CE-associated vasculotoxicity, and propose to demonstrate the role of cations in CE-associated vasculotoxicity. Aim #1: CE exposure in human umbilical vein endothelial cells (HUVEC) induces pro-inflammatory activation and permeability. HUVEC monolayers exposed to CE or saline will be analyzed for CE (Gas Chromatography-Mass Spectrometry), and electrolyte (electrolyte analyzers) and oxygen/carbon dioxide levels (gas analyzers). Fixed monolayers will be stained for counting of intercellular gap formations (silver stain), and surface markers of activation (immunohistochemistry). Aim #2: CE exposure in HUVEC activates pro-inflammatory signaling/gene expression pathways. CE or saline treated monolayers will be tested for RNA and protein expression and activity changes utilizing electrophoretic mobility shift assay and supershift, RNAse protection assay, and Western blot. Targets are signaling and activation markers, and include Nuclear Factor kB, interleukin-8, and Vascular Cell Adhesion Molecule-1. Aim #3: HUVEC activation after exposure to CE is associated with high sustained cytoplasmic cation flux. Monolayers of HUVEC will be treated with CE and intracellular cation flux (calcium, magnesium, and hydronium) recorded utilizing fluorescence spectrometry. Flow cytometry and/or Western blotting will be used to monitor changes in calcium channel density over time. The proposed Aims will provide further insight into the pathogenic sequelae resulting from production of CE in humans.